JP7037198B2 - Prophylactic and / or therapeutic agents for autoimmune diseases, as well as vaccines - Google Patents

Description

The present invention relates to prophylactic and / or therapeutic agents for autoimmune diseases and vaccines for the prevention and / or treatment of autoimmune diseases.

The immune system originally exists as a defense mechanism of a living body against the invasion of harmful foreign substances from the outside world. However, sometimes this function of the immune system is harmful to the living body as a result. It is known that a living body causes an immune response not only against foreign substances from the outside world but also against its own components, which is called an autoimmune phenomenon. A disease in which a certain condition is caused by autoimmunity is called an autoimmune disease.

Autoimmune diseases are systemic diseases, but there are two major types: organ-specific diseases (organ-specific autoimmune diseases) and non-organ-specific diseases (organ-non-specific autoimmune diseases). Be separated. Many of these autoimmune diseases have tissue lesions and are associated with tissue damage.

Systemic lupus erythematosus (SLE), an organ-nonspecific autoimmune disease, is known to be due to increased production of autoantibodies such as anti-dsDNA antibodies, but molecular biological studies of the processes that cause SLE. , Elucidation of the relationship with various pathological conditions, and development of treatment methods are expected.

When the experimental animal is repeatedly immunized with the same antigen, the immune response reaches the extreme stage and eventually becomes exhausted. As a result, it is known that various autoimmune diseases (pathological conditions) accompanied by tissue injury occur (see, for example, Patent Document 1 and Non-Patent Document 1).

Research on tissue damage caused by autoimmune diseases is currently underway, and it has been clarified that the cross-presentation of antigens by antigen-presenting cells is involved in tissue damage. Specifically, (1) suffering from autoimmune disease enhances antigen cross-presentation in dendritic cells, and (2) enhances cross-presentation in dendritic cells activates CD8 ⁺ T cells. , It has been clarified that the CD8 ⁺ T cells induce cell damage (see, for example, Patent Document 2 and Non-Patent Document 1).

By repeating immune manipulation with antigens against mice, the present inventors have created a new type of T cells called autoantibody-inducing CD4 T cells (aiCD4 T cells). So far, aiCD4 T cells, which are produced in the periphery, induce various autoantibodies and mature cytotoxic T cells (CTLs) to cause various organ damages and cause SLE. (Non-Patent Document 1), and further, that aiCD4 T cells are present in the CD4 T cell subpopulation of PD-1 ⁺ CD45RB ^low 122 ^low (PD-1 ⁺ CD45RB ^lo 122 ^lo ), this cell subpopulation. It has been found that when the cells are adopted into naive mice, autoantibodies are observed in the sera of recipient mice 2 weeks after the transfer (see Non-Patent Document 2).

Furthermore, the present inventors have found a marker for more easily identifying a cell that causes the onset of an autoimmune disease, and based on this, a technique that enables objective and accurate diagnosis of an autoimmune disease. It has been successfully developed (see Patent Document 3).

Japanese Patent Publication "Japanese Patent Laid-Open No. 2006-288382 (published October 26, 2006)" Japanese Patent Publication "Japanese Patent Laid-Open No. 2010-004750 (Published January 14, 2010)" Japanese Patent Publication "Japanese Patent Laid-Open No. 2017-003329 (published January 5, 2017)"

Tsumiyama K. et al., PLoS ONE, Vol.4, No.12, e8382, 2009 Miyazaki Y. et al., The Journal of Immunology vol.192, no.2, supplement 177.7, 2014 Zhang Q et al., N Engl J Med 361 (21): 2046-2055, 2009 Randall KL et al., Nat Immunol 10 (12): 1283-1291, 2009 Randall KL et al. Disease Markers 29: 141-150, (2010) Randall KL et al., J Exp Med 208 (11): 2305-2320, 2011 Werner M and Jumaa H, Nat Immunol 13 (6): 525-526, 2012 Jabara HH et al., Nat Immunol 13 (6): 612-620, 2012

However, the drugs used for the prevention and / or treatment of autoimmune diseases are not yet sufficient, and further development of new drugs has been desired.

It is an object of the present invention to provide a novel agent used for the prevention and / or treatment of autoimmune diseases.

As a result of diligent studies, the present inventor has found that the symptoms of autoimmune diseases can be improved by using an anti-DOCK8 antibody, and has completed the present invention. The findings found by the present inventor suggest that the DOCK8 protein is involved in the pathogenic process of autoimmune diseases, which is a surprising finding for the field.

In order to solve the above problems, the prophylactic and / or therapeutic agent for an autoimmune disease according to one aspect of the present invention is characterized by containing an anti-DOCK8 antibody as an active ingredient.

In order to solve the above-mentioned problems, the vaccine for preventing and / or treating an autoimmune disease according to one aspect of the present invention is a DOCK8 protein, a partial sequence of the DOCK8 protein, or a partial sequence of the DOCK8 protein or the DOCK8 protein. It is characterized by containing the expression vector of the above as an active ingredient.

According to one aspect of the present invention, autoimmune diseases can be prevented and / or treated.

It is a graph which shows the test result of proteinuria in the Example of this invention. It is an image which shows the test result about the localization of DOCK8 protein in the Example of this invention.

An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments and examples can be used. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. In addition, all the academic and patent documents described in the present specification are incorporated as references in the present specification. Further, unless otherwise specified in the present specification, "AB" representing a numerical range is intended to be "A or more and B or less".

[1. Preventive and / or therapeutic agents for autoimmune diseases]
As described above, the present inventors have so far indicated an increase in CD4 T cells expressing dedicator of cytokinesis protein 8 (hereinafter, DOCK8) (DOCK8-positive CD4 T cells (also referred to as DOCK8 ⁺ CD4 T cells)). (Patent Document 3) reports a technique for diagnosing autoimmune diseases. However, it was unclear by what mechanism these DOCK8 ⁺ CD4 T cells were involved in the pathogenic process of autoimmune diseases. Therefore, as a result of diligent studies by the present inventors, it was surprisingly found that the antibody against DOCK8 has a preventive / therapeutic effect on autoimmune diseases. It should be noted that even if the presence of DOCK8 ⁺ CD4 T cells can be one of the diagnostic indicators for autoimmune diseases, there is a very large gap in the development of therapeutic agents.

The prophylactic and / or therapeutic agent for autoimmune diseases of the present embodiment contains an anti-DOCK8 antibody as an active ingredient.

As used herein, the term "autoimmune disease" means that an autoantibody (eg, anti-Sm antibody, anti-dsDNA antibody, rheumatoid factor (RF)) against a self-component (eg, immunoglobulin) is detected and autoimmune. Intended for diseases associated with the pathology. Autoimmune diseases include organ-specific autoimmune diseases (eg, chronic thyroiditis, primary mucosal edema, thyroid poisoning, malignant anemia, Good Pasture syndrome, acute progressive glomerulonephritis, severe myasthenia, vulgaris vulgaris). Blisters, vesicular vesicles, insulin-resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastric inflammation, male infertility, premature climacteric, crystalline venous inflammation, sympathitis, polysclerosis, Ulceral colitis, primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemochromatosis, idiopathic thrombocytopenic purpura, and Sjogren's syndrome), and organ-nonspecific self Immune diseases such as rheumatoid arthritis, systemic erythematosus (SLE), discoid erythematosus, polymyopathy, scleroderma, and mixed connective tissue disease, etc.). Rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma and mixed connective tissue disease are known as collagen diseases. That is, collagen disease is included in systemic autoimmune diseases. The autoimmune disease in one embodiment of the present invention is preferably collagen disease, more preferably systemic lupus erythematosus.

The DOCK8 protein is a member of the DOCK family protein and is a protein having a molecular weight of 238 kDa having the function of a guanine nucleotide exchange factor (GEF). The DOCK8 protein is known to regulate the polymerization of the actin cytoskeleton through activation of the Rho family small G proteins. The rearrangement of the actin cytoskeleton is involved in various cell functions such as cell differentiation / proliferation, migration, and signal transduction.

In recent years, a number of immunological research results on DOCK8 have been reported, triggered by the discovery that a deficiency in the human DOCK8 gene causes severe combined immunodeficiency disease (see Non-Patent Documents 3 to 8).

In the present specification, "DOCK8" is intended to be both a DOCK8 protein and a DNA (gene) encoding the DOCK8 protein, unless otherwise specified.

The anti-DOCK8 antibody is one that binds to the DOCK8 protein or a partial sequence of the DOCK8 protein. At this time, the origin of the DOCK8 protein to be bound is not particularly limited, and examples thereof include mammals. Examples of mammals include mice, rats, rabbits, goats, monkeys and humans, preferably mice, rats and humans, and more preferably humans.

In the examples described later, it is suggested that the DOCK8 protein present on the surface of the cell membrane is involved in the pathogenic mechanism of SLE. Therefore, the anti-DOCK8 antibody according to the present embodiment may be an antibody that inhibits the localization of the DOCK8 protein to the cell membrane. For example, each of cell A in which the DOCK8 protein is localized in the cell membrane and the cell B to which an arbitrary antibody is added is immunostained with an anti-DOCK8 antibody, and the staining intensity in the vicinity of the cell membrane in the cell B is increased. If the staining intensity in the vicinity of the cell membrane in cell A is lower than that of the cell membrane, it can be determined that the arbitrary antibody is an antibody that inhibits the localization of the DOCK8 protein to the cell membrane.

More specifically, the anti-DOCK8 antibody is a human-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5, a mouse-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, or a polypeptide thereof. It may be one that binds to a partial sequence of.

The number of amino acids constituting the partial sequence is not particularly limited, and for example, 400 or less, 350 or less, 300 or less, 200 or less, 100 or less, 90 or less, 80 or less, 70 or less, It may be 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. The lower limit of the number of amino acids constituting the partial sequence is not particularly limited, and is, for example, 3, 4, 5, 6, 7, 8, 9, or 10. You may.

The anti-DOCK8 antibody is a DOCK8 protein or an antibody that specifically binds to a partial sequence of the DOCK8 protein. It should be noted that, in the present specification, an antibody "specifically binds" to a DOCK8 protein or a partial sequence of a DOCK8 protein means that the antibody has a DOCK8 protein or DOCK8 rather than to other polypeptides. It is intended to bind with high affinity to partial sequences of proteins. Here, "high affinity" means, for example, that the binding constant (Ka: binding constant) is 10 ⁷ M ^-1 or more, preferably 10 ⁸ M ^-1 or more, more preferably 10 ⁹ M ^-1 or more, and more preferably. Is intended for affinity of 10 ¹⁰ M ^-1 or higher, more preferably 10 ¹¹ M ^-1 or higher, more preferably 10 ¹² M ^-1 or higher, and most preferably 10 ¹³ M ^-1 or higher.

The anti-DOCK8 antibody may be an entire anti-DOCK8 antibody, an entire anti-DOCK8 antibody, an anti-DOCK8 antibody fragment, or an anti-DOCK8 antibody fragment. For example, the DOCK8 antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or a fragment thereof (for example, F (ab') ₂ , Fab', Fab, or Fv). good. For example, if the anti-DOCK8 antibody is a monoclonal antibody, it is possible to reduce the non-specific binding of the anti-DOCK8 antibody to a target other than the DOCK8 protein, and thus the prevention and / or prevention of autoimmune diseases of the present embodiment. The pharmacological effect of the therapeutic agent can be enhanced, and the prevention of the autoimmune disease of the present embodiment and / or the occurrence of side effects of the therapeutic agent can be suppressed. The anti-DOCK8 antibody may be IgA, IgD, IgE, IgG, IgM, or a fragment thereof.

Anti-DOCK8 antibodies are described by well-known methods (eg, (1) HarLow et al., "Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York (1988)", and (2) Iwasaki et al., "Single clone antibody hybridoma". It can be produced according to ELISA, Kodansha (1991) "). Of course, it is also possible to use a commercially available antibody as the anti-DOCK8 antibody.

Monoclonal antibodies are used by methods well known in the art (eg, (1) hybridoma method (Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), (2) trioma method, (3) human. According to the B-cell hybridoma method (Kozbor, Immunology Today 4,72 (1983)) and (4) Monoclonal Antibodies and Cancer (Alan R Liss, Inc., 77-96). Can be made.

Monoclonal antibodies can also be produced according to the phage display method. As a method for producing a monoclonal antibody by the phage display method, a known method may be used. An example of a method for producing a monoclonal antibody by the phage display method will be described below. First, mRNA is prepared from an animal immunized with the DOCK8 protein, cDNA is prepared using the mRNA as a template, and a single-chain antibody (scFV) gene encoding only the variable region of the antibody is prepared from the cDNA. Next, the single-stranded antibody gene is inserted into a phagemid vector, the phagemid vector is introduced into Escherichia coli, and then the E. coli is infected with the phage. This allows the scFV antibody to be expressed on the phage capsule. An anti-DOCK8 monoclonal antibody can be prepared by screening an scFV antibody expressed on the phage capsule that binds to the DOCK8 protein. Preparation of mRNA, preparation of cDNA, insertion of a single-stranded antibody gene into a phagemid vector, introduction of a phagemid vector into Escherichia coli, infection of a phage, and screening of an antibody may be carried out according to a well-known method.

Chimeric antibodies are intended as antibodies in which the constant region is replaced by the constant region of a human antibody. Chimeric antibodies can be made according to well-known methods (see, eg, European Patent Publication EP0125023).

The humanized antibody is intended to be an antibody in which the structure of a human antibody is replaced by other than the complementarity determining regions (CDRs) of the H chain and the L chain.

The human antibody is intended to be an antibody produced using a transgenic animal into which a gene involved in human antibody production has been introduced (see, for example, EP0546073).

F (ab') ₂ , Fab', Fab, and Fv can be obtained by degrading the entire antibody with a protease (eg, papain or pepsin) and, if necessary, further reducing the degradation product. Can be done. In addition, F (ab') ₂ , Fab', Fab, and Fv isolate these cDNAs from hybridomas that produce antibodies, insert the cDNAs into expression vectors, and introduce the expression vectors into the host. Can be obtained by In this case, the antibody fragment can also be obtained as a fusion protein of the antibody fragment and another protein.

The amount of the anti-DOCK8 antibody contained in the prophylactic and / or therapeutic agent for autoimmune disease of the present embodiment is not particularly limited, and is, for example, 0 when the prophylactic and / or therapeutic agent is 100% by weight. .001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, 0.1% by weight to It may be 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to 70% by weight. It may be 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight, 0. It may be 1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight.

The amount of the anti-DOCK8 antibody contained in the prophylactic and / or therapeutic agent for the autoimmune disease of the present embodiment is not particularly limited, and is, for example, 0.01 mg to 1000 mg, 0.1 mg to 1000 mg, 1 mg to 1000 mg, and the like. It may be 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg.

The prophylactic and / or therapeutic agent for autoimmune diseases of this embodiment may contain components other than the anti-DOCK8 antibody (eg, pharmaceutically acceptable carriers). Examples of the component other than the anti-DOCK8 antibody include excipients, lubricants, binders and disintegrants when the prophylactic and / or therapeutic agent for the autoimmune disease of the present embodiment is a solid preparation. When the preventive and / or therapeutic agent for the autoimmune disease of the present embodiment is a liquid preparation, the solvent, the solubilizing agent, the suspending agent, the tonicity agent, the buffering agent and the soothing agent can be mentioned. Can be done. In addition, as components other than the anti-DOCK8 antibody, preservatives, antioxidants and stabilizers can also be mentioned.

Examples of the above-mentioned "excipient" include, but are not limited to, lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch and crystalline cellulose.

Examples of the above-mentioned "lubricating agent" include, but are not limited to, magnesium stearate, calcium stearate, wax, talc and colloidal silica.

Examples of the above-mentioned "binder" include, but are not limited to, pregelatinized starch, methyl cellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and polyvinylpyrrolidone.

Examples of the above-mentioned "disintegrant" include, but are not limited to, starch, carboxymethyl cellulose, low-degree-of-substitution hydroxypropyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium and carboxymethyl starch sodium.

Examples of the "solvent" include, but are not limited to, water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and tricaprylin.

Examples of the above-mentioned "solubilizing agent" include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. Not limited to these.

Examples of the above-mentioned "suspending agent" include surfactants (for example, stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate) and highly hydrophilic. Molecules (eg, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose) are not limited thereto.

Examples of the above-mentioned "isotonic agent" include, but are not limited to, sodium chloride, glycerin and D-mannitol.

Examples of the above-mentioned "buffering agent" include, but are not limited to, phosphates, acetates, carbonates and citrates.

Examples of the above-mentioned "painless agent" include, but are not limited to, benzyl alcohol.

Examples of the above-mentioned "preservative" include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid.

Examples of the above-mentioned "antioxidant" include, but are not limited to, sulfites and ascorbic acid.

The above-mentioned "stabilizer" may be any one usually used in the pharmaceutical field and is not particularly limited.

The amount of components other than the anti-DOCK8 antibody contained in the prophylactic and / or therapeutic agent for autoimmune diseases of the present embodiment is not particularly limited, and for example, when the prophylactic and / or therapeutic agent is 100% by weight. In addition, it may be 0% by weight to 99.99% by weight, 0% by weight to 99.99% by weight, 0% by weight to 99.9% by weight, or 5% by weight. It may be up to 99.9% by weight, 10% by weight to 99.9% by weight, 20% by weight to 99.9% by weight, and 30% by weight to 99.9% by weight. %, 40% by weight to 99.9% by weight, 50% by weight to 99.9% by weight, 60% by weight to 99.9% by weight. It may be 70% by weight to 99.9% by weight, 80% by weight to 99.9% by weight, or 90% by weight to 99.9% by weight.

The amount of components other than the anti-DOCK8 antibody contained in the preventive and / or therapeutic agent for autoimmune diseases of the present embodiment is not particularly limited, and is, for example, 0.01 mg to 1000 mg, 0.1 mg to 1000 mg, and 1 mg. It may be ~ 1000 mg, 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg.

The method for administering the prophylactic and / or therapeutic agent for the autoimmune disease of the present embodiment is not particularly limited and may be orally administered, or parenterally, intravenously, intrarectally, intraperitoneally, or muscle. It may be administered intranasally, intranasally or subcutaneously.

The dosage form of the preventive and / or therapeutic agent for autoimmune disease of the present embodiment is not particularly limited, and is, for example, an injection (for example, intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, muscle). It may be an injection (or a drip injection), a nasal drop, or a suppository.

[2. Vaccines for the prevention and / or treatment of autoimmune diseases]
As shown in the examples described below, anti-DOCK8 antibodies can prevent and / or treat autoimmune diseases. This indicates that vaccines containing substances (eg, antigens) that can be administered to the body to induce the production of anti-DOCK8 antibodies can also prevent and / or treat autoimmune diseases. There is.

Therefore, the vaccine for the prevention and / or treatment of autoimmune diseases of the present embodiment contains the expression vector of the DOCK8 protein, the partial sequence of the DOCK8 protein, or the partial sequence of the DOCK8 protein or the DOCK8 protein as an active ingredient. is doing.

For example, the vaccine for the prevention and / or treatment of an autoimmune disease of the present embodiment may be a DOCK8 protein derived from a species different from the subject, a partial sequence of the DOCK8 protein derived from a species different from the subject, or , A DOCK8 protein derived from a species different from the administration target or an expression vector of a partial sequence of the DOCK8 protein may be contained as an active ingredient.

The vaccine for the prevention and / or treatment of autoimmune diseases of the present embodiment and the above-mentioned prophylactic and / or therapeutic agent for autoimmune diseases differ only in the substance used as an active ingredient, and have the same other configurations. There may be. Since the vaccines for the prevention and / or the treatment of autoimmune diseases of the present embodiment have already been described except for the active ingredient, the description thereof will be omitted here.

The DOCK8 protein, which is an active ingredient, and the partial sequence of the DOCK8 protein include a human-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5, and a mouse-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. , And partial sequences of these polypeptides.

The number of amino acids constituting the partial sequence is not particularly limited, and for example, 400 or less, 350 or less, 300 or less, 200 or less, 100 or less, 90 or less, 80 or less, 70 or less, It may be 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. Further, the partial sequence may be a partial sequence in the DOCK8 protein, and the position in the DOCK8 protein is not limited.

The DOCK8 protein and the partial sequence of the DOCK8 protein can be prepared by a known method. For example, (i) it can be prepared using a known peptide synthesizer, or (ii) a DOCK8 protein and a cDNA encoding a partial sequence of the DOCK8 protein are inserted into an expression vector, and the expression vector is inserted. It can also be produced by introduction into a desired host (eg, E. coli, yeast, etc.).

The expression vector of the DOCK8 protein or the partial sequence of the DOCK8 protein as the active ingredient is one in which the cDNA encoding the DOCK8 protein or the partial sequence of the DOCK8 protein is expressively inserted into the vector.

Specific examples of the vector are not particularly limited, and are, for example, a plasmid, a viral vector (for example, a vaccinia virus vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector), or a virus. Particles can be mentioned. The specific example of the promoter inserted into the vector is not particularly limited, and may be appropriately selected depending on the above-mentioned vector.

A DOCK8 protein or a cDNA encoding a partial sequence of the DOCK8 protein is inserted into the vector in an expressible manner. The specific base sequence of the cDNA is not particularly limited. The cDNA encoding the DOCK8 protein or the partial sequence of the DOCK8 protein includes a human-derived polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3, a mouse-derived polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, and a polynucleotide derived from a mouse. , Partial sequences of these polynucleotides can be mentioned.

The amount of the active ingredient contained in the preventive and / or therapeutic vaccine of the present embodiment is not particularly limited, and for example, the preventive and / or therapeutic vaccine is 100% by weight. In this case, it may be 0.001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, or 0. .1% by weight to 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to It may be 70% by weight, 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight. It may be 0.1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight.

The amount of the anti-active ingredient contained in the vaccine for the prevention and / or treatment of the autoimmune disease of the present embodiment is not particularly limited, and is, for example, 0.01 mg to 1000 mg, 0.1 mg to 1000 mg, 1 mg. It may be ~ 1000 mg, 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg.

One aspect of the present invention can also be configured as follows.

The prophylactic and / or therapeutic agent for an autoimmune disease according to one aspect of the present invention is characterized by containing an anti-DOCK8 antibody as an active ingredient.

In the prophylactic and / or therapeutic agent for an autoimmune disease according to one aspect of the present invention, the autoimmune disease is preferably systemic lupus erythematosus (SLE).

In the prophylactic and / or therapeutic agent for autoimmune diseases according to one aspect of the present invention, the anti-DOCK8 antibody is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2 or 5, or a partial sequence of the polypeptide. It is preferable that it binds to.

In the prophylactic and / or therapeutic agent for autoimmune diseases according to one aspect of the present invention, the anti-DOCK8 antibody is preferably a monoclonal antibody.

The vaccine for the prevention and / or treatment of an autoimmune disease according to one aspect of the present invention contains an expression vector of a DOCK8 protein, a partial sequence of the DOCK8 protein, or a partial sequence of the DOCK8 protein or the DOCK8 protein as an active ingredient. It is characterized by being.

[Test 1]
BALB / c mice (Charles River Japan Inc.) were repeatedly intraperitoneally administered with 0.5 mg of ovalbumin (OVA: grade V; Sigma) or PBS every 5 days. As a result, mice exhibiting symptoms of autoimmune disease were produced.

24 hours before the 6th, 9th and 12th OVA administrations and the 6th, 9th and 12th PBS administrations, 50 μg of anti-DOCK8 antibody derived from rabbit or 50 μg of control IgG (rabbit IgG). Was intraperitoneally administered to BALB / c mice.

As the anti-DOCK8 antibody, a commercially available anti-DOCK8 polyclonal antibody (proteintech, DOCK8 Polyclonal Antibody, Catalog No. 11622-1-AP, antigen: polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 5) was used. ..

After the 12th OVA administration and after the 12th PBS administration, proteinuria (proteinuria), which is an indicator of the onset of nephritis in systemic lupus erythematosus, by a color reaction using Arbusticus (registered trademark) (SIEMENS). proteinuria) was detected. In addition, HE (Hematoxylin-Eosin) staining was performed according to a well-known method, and pathological evaluation of the kidney was performed according to the WHO classification. Specifically, the renal symptoms observed according to the criteria described in "Weening JJ et al., The classification of glomerulonephritis in systemic lupus erythematosus revised. J Am Soc Nephrol 15 (2): 241-250, 2004" , Class I-II, Class III, Class IV, and Class V, and pathological evaluation of the kidney was performed.

FIG. 1 shows the test results of proteinuria, and Table 1 below shows the pathological evaluation of the kidney according to the WHO classification.

As shown in FIG. 1, proteinuria was detected in the BALB / c mouse group (“OVA × 12 + control IgG” in FIG. 1) to which control IgG was administered before the 6th, 9th, and 12th OVA administrations. Was done. On the other hand, in the BALB / c mouse group (“OVA × 12 + anti-DOCK8 Ab” in FIG. 1) to which the anti-DOCK8 antibody was administered before the 6th, 9th and 12th OVA administrations, the level of proteinuria decreased. did. In FIG. 1, "PBS x 12 + control IgG" is a test result of a BALB / c mouse group to which control IgG was administered before the 6th, 9th, and 12th administration of PBS.

In addition, as shown in Table 1, the BALB / c mouse group (“OVA × 12 + control IgG” in Table 1) to which control IgG was administered before the 6th, 9th, and 12th OVA administrations was classified as WHO. Severe nephritis such as type IV and type V was detected in. On the other hand, in the BALB / c mouse group (“OVA × 12 + anti-DOCK8 Ab” in Table 1) to which the anti-DOCK8 antibody was administered before the 6th, 9th and 12th OVA administrations, nephritis was almost completely suppressed. Was done. In Table 1, "PBS x 12 + control IgG" is the test result of the BALB / c mouse group to which control IgG was administered before the 6th, 9th and 12th administration of PBS.

〔試験２〕
ＢＡＬＢ／ｃマウス（Charles River Japan Inc.）に、卵白アルブミン（ＯＶＡ：grade V; Sigma）０．５ｍｇを、５日毎に繰り返し腹腔内投与した。これによって、自己免疫疾患の症状を呈するマウスを作製した。

[Test 2]
BALB / c mice (Charles River Japan Inc.) were repeatedly intraperitoneally administered 0.5 mg of ovalbumin (OVA: grade V; Sigma) every 5 days. As a result, mice exhibiting symptoms of autoimmune disease were produced.

Twenty-four hours before the sixth, ninth and twelfth administration of OVA, 50 μg of an anti-DOCK8 antibody derived from rabbit (the same antibody used for [Test 1]) or 50 μg of control IgG (rabbit IgG) was added. It was administered intraperitoneally to BALB / c mice.

Blood was collected after the 12th OVA administration, and autoantibodies (specifically, RF (rheumatoid factor), anti-Sm Ab, and anti-dsDNA Ab) in serum were measured by ELISA (enzyme-linked immunosorbent assay). Detected.

Table 2 below shows the ELISA test results.

In addition, as shown in Table 2, compared with the BALB / c mouse group (“OVA × 12 + control IgG” in Table 2) to which control IgG was administered before the 6th, 9th, and 12th OVA administrations. , All three types of autoantibodies in the BALB / c mouse group (“OVA × 12 + anti-DOCK8 Ab” in FIG. 3) to which the anti-DOCK8 antibody was administered before the 6th, 9th and 12th OVA administrations. However, it decreased significantly.

〔試験３〕
ＢＡＬＢ／ｃマウス（Charles River Japan Inc.）に、卵白アルブミン（ＯＶＡ：grade V; Sigma）０．５ｍｇを、５日毎に繰り返し腹腔内投与した。これによって、自己免疫疾患の症状を呈するマウスを作製した。

[Test 3]
BALB / c mice (Charles River Japan Inc.) were repeatedly intraperitoneally administered 0.5 mg of ovalbumin (OVA: grade V; Sigma) every 5 days. As a result, mice exhibiting symptoms of autoimmune disease were produced.

After the 12th OVA administration, T cells were fractionated into several subpopulations. Cytoplasmic fractions and membrane fractions were obtained from T cells of each subpopulation according to a well-known method.

A cytoplasmic fraction of 10 μg per lane and a membrane fraction of 10 μg per lane were subjected to electrophoresis (10% polyacrylamide gel), and the proteins contained in each fraction were separated. The DOCK8 protein contained in the separated protein was detected by Western blotting using an anti-DOCK8 antibody. The results are shown in FIG.

In FIG. 2, lanes 1 and 5 are test results of cytoplasmic and membrane fractions derived from Whole T cells, respectively. In addition, lanes 2 and 6 are the test results of the cytoplasmic fraction and the membrane fraction derived from CD45RB ^high 122 ^high cells, respectively. In addition, lanes 3 and 7 are the test results of the cytoplasmic fraction and the membrane fraction derived from PD-1 ^- CD45RB ^low 122 ^low cells, respectively. In addition, lanes 4 and 8 are the test results of the cytoplasmic fraction and the membrane fraction derived from PD-1 ⁺ CD45RB ^low 122 ^low cells, respectively.

Among these cells, PD-1 ⁺ CD45RB ^low 122 ^low cells contain autoantibody-inducing CD4 T cells (aiCD4 T cells). Also, in FIG. 2, the image observed at a position of approximately 230 kDa corresponds to the DOCK8 protein.

As is clear from lanes 4 and 8 of FIG. 2, in PD-1 ⁺ CD45RB ^low 122 ^low cells, not only the cytoplasmic fraction but also the membrane fraction contained a large amount of DOCK8 protein. This indicates that in D-1 ⁺ CD45RB ^low 122 ^low cells, many DOCK8 proteins are localized on the surface of the cell membrane. Since PD-1 ⁺ CD45RB ^low 122 ^low cells can be recognized by flow cytometry using a DOCK8 antibody, many DOCK8 proteins are localized on the surface of the cell membrane in D-1 ⁺ CD45RB ^low 122 ^low cells. It is thought that it is.

From previous studies, aiCD4 T cells contained in the CD4 T cell subpopulation (specifically, PD-1 ⁺ CD45RB ^low 122 ^low ) induce various autoantibodies and cytotoxic T cells (CTL). ) Is matured to cause various organ disorders and cause SLE. Therefore, the results of this test suggest that not only the DOCK8 protein present in the cytoplasmic fraction but also the DOCK8 protein present on the surface of the cell membrane is involved in the pathogenic mechanism of SLE.

One aspect of the invention can be used for the prevention and / or treatment of autoimmune diseases.

Claims (3)

A prophylactic and / or therapeutic agent for systemic lupus erythematosus (SLE) , which comprises an anti-DOCK8 antibody as an active ingredient. The whole body according to claim 1 , wherein the anti-DOCK8 antibody binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2 or 5, or a partial sequence of the polypeptide. Prophylactic and / or therapeutic agents for sex lupus erythematosus (SLE) . The prophylactic and / or therapeutic agent for systemic lupus erythematosus (SLE) according to claim 1 or 2 , wherein the anti-DOCK8 antibody is a monoclonal antibody.

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